Insulin resistance in adipose tissue and muscle is a characteristic feature of patients with type 2 diabetes. Insulin resistance precedes the development of diabetes, and is considered to be a key pathogenetic factor. The molecular mechanisms responsible for insulin resistance are largely unknown. However, one of the possibilities is that a key protein in insulin receptor signaling, IRS-1, that is normally tyrosine phosphorylated in response to insulin, may be desensitized by serine phosphorylation. The aims of this proposal are: 1) 1. To determine whether IRS-1 is phosphorylated on serine residues at positions 616, 636, and 666 in situ in isolated rat adipocytes; 2) To determine whether insulin stimulation of phosphorylation of IRS-1 at Ser616,636,666 is mediated by insulin signaling through the MAP kinase or PI 3-kinase pathways; and 3) To determine if insulin resistance is associated with increased IRS-1616,636,666 phosphorylation. I will use rat adipocytes incubated in primary culture with TNF-alpha or insulin to induce insulin resistance and test the hypothesis that IRS-1 Ser616,636,666 phosphorylation is increased in insulin resistant adipocytes. To accomplish these aims, primary cultures of rat adipocytes will be used together with adenovirally expressed IRS-1 proteins to determine the regulation of phosphorylation of serine residues 616,636, and 666 in situ.